Herstellung und Anwendung von hochkomplexen Peptidbibliotheken

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Translation of abstract (English)

Proteins ability to physically interact with other proteins or biomolecules provides the essential basic of protein function in human organisms. Interferences of these interactions can result in male-function of engaged signal transduction and metabolic pathways finally causing diseases and death. Protein interactions have to be studied in detail to understand molecular basis of involved processes. In addition to genetic, cell biological and complex biochemical approaches array-technologies become more important for systematically identification of protein interaction partners. Usage of such protein and peptide arrays is still limited by low availability. Peptide arrays, which are appropriate tools for high-throughput-screenings of agonists and antagonists and therefore for development of drugs, are commercially generated by established SPOT-synthesis techniques resulting in array densities of 25 peptides per cm2. Therefore, at the German Cancer Research Center (Heidelberg/Germany) a novel method was developed to synthesise peptide arrays with higher complexities and low expenditures of time and material resulting in significant reduced manufacturing costs. This technique is based on solid amino acid particles whereby the particles consist of one pre-activated L-amino acid and several other additives embedded in a solid matrix. These particles can be addressed to defined reaction regions of a solid supports by either a custom-built laser printer or specific microchips. Afterwards by heating the support the particle matrix liquefies and initiates coupling of the amino acids onto chemical modified supports. Repetitive addressing, coupling and washing cycles finally result in the combinatorial synthesis of a peptide array. This novel method outperforms established synthesis techniques regarding their complexity of 400 peptide spots per cm2 in case of laser printing and 10,000 – 40,000 peptide spots using the microchip approach whereby production costs were minimized. Prior to establishing this novel synthesis method peptide arrays should be comprehensively characterized and usefulness evaluated in different model applications. Specific peptide substrates of different kinases e.g. cAMP-dependent protein kinase (PKA) and c-Src were combinatorially synthesized and phosphorylated. In accordance with literature the specific phosphorylation of serine embedded in the recognition sequence RRXS (X = variant amino acid) by cAMP-dependent protein kinase could be detected. This example proofed the usefulness of the novel peptide array synthesis using solid amino acid particles for the identification of specific kinase substrates. Experiments for the identification of catalytic active metallopeptides are based on results achieved by Berkessel et al., who identified specific metal-peptide complexes and metal binding peptide derivates catalyzing hydrolytical cleavage of phosphate esters and phosphodiesters. Despite intensive experiments all attempts were in vain to apply this approach to the synthesized peptide arrays. The stained reaction products were not detected unambiguously, which is why specific catalytic activity of tested metallopeptides could not be verified. Further model approaches yield in identification of metal binding peptides, which can be used in nuclear medicine. Radiopharmaceuticals often contain the metastable 99mTechnetium bound to a bioactive molecule as organometallic complex, which recognizes a target structure. For the identification of new [99mTc(H2O)3(CO)3]+-binding peptides, a hexapeptide library with 133,224 peptide spots was generated on a chemically modified glass support (21 x 22 cm) with the laser printer. Although the first and last amino acids of the hexapeptides were not identified and final verification of results (e.g. HPLC) is not completed by now, specific peptide candidates were identified, which bind [99mTc(H2O)3(CO)3]+ or -as a matter of reference- 99mTcO3+ under given reaction conditions. The results provide evidence for the usefulness of our new peptide arrays synthesized via a laser printer for the development of medical relevant metal-peptide complexes. In a further proof-of-concept experiment the essential feasibility of combinatorial synthesis on a microchip was documented by simple immunoassaying.